Lipase-Catalyzed Production of Solid Fat Containing Conjugated Linoleic Acid in Binary Models

Food Sci. Biotechnol. Vol. 18, No. 3, pp. 803 ~ 807 (2009)

RESEARCH NOTE

Xue-Mei Zhu, Md. Abdul Alim, Jiang-Ning Hu, Prakash Adhikari, Jeung-Hee Lee, and Ki-Teak Lee* Department of Food Science and Technology, Chungnam National University, Daejeon 305-764, Korea

Abstract Solid fats were esterified with solid phase of rice bran oil (S-RBO), palm stearin (PS), and conjugated linoleic acid (CLA) at 2 substrate mole ratios (S-RBO:PS:CLA of 1:1:2 and 1:3:4). The major fatty acids were palmitic, oleic, and CLA in 36 hr products. The solid fat content (SFC) of the 1:1:2 product was 12.8% while the SFC of 1:3:4 product was 45.1% at 20oC. The SFCs after 20oC reduced when the reaction time increased from 1 to 36 hr, suggesting that the change of triacylglycerol species was augmented by extending reaction time. Keywords: rice bran oil, palm stearin, conjugated linoleic acid, enzymatic esterification, solid fat content

Introduction of natural antioxidants such as tocopherol, oryzanol, and tocotrienol (12-14). Also it is known that RBO reduces the Conjugated linoleic acid (CLA) refers to the mixture of harmful low density lipoproteins (LDL) cholesterol (15). positional and geometric isomers of linoleic acid with As a useful approach, fractionation could alter the melting conjugated double bonds. The nutritional and healthy point and solidify characteristics by enriching the fat or benefits of CLA have been widely studied. Lee et al. (1) fatty acids. Fractionation was successfully used by Yu et al. investigated the effect of CLA on retardation of (16) to selectively enrich the content of unsaturated fatty atherosclerosis. Besides, Chin et al. (2) found the activity acid (UFA) by fractionation with acetone at various of CLA on enhancement of growth, and O’Shea et al. (3) temperatures from RBO. Also high saturated fatty acids proved CLA could increase of immunity. Additionally, (SFA) were enriched from RBO by Alim et al. (17). In effect of reducing body weight of CLA was reported by their results, CLA was incorporated to the solid phase of Park et al. (4). Therefore, supplement of CLA into foods RBO (S-RBO) and PS to prepare nutritional solid fat by attracts considerable attention due to these pharmaceutical enzyme-catalyzed esterification, in which response surface and nutritional values. methodology was used to optimize the reaction. However, One feasible approach to increase the CLA amount in the rate of incorporation of CLA was relatively low foods is transesterification of CLA with triacylglycerols (10.9%). Therefore, in this study, reaction time as the (TAG) by chemical or enzymatic catalysts. Through single factor was investigated to raise the incorporation incorporation of a required acyl group into a specific amount of CLA in solid fat products from binary model of position of TAG, lipase-catalyzed esterification provides a substrate molar ratios. Two substrate ratios were selected useful way to improve the nutritional properties of lipids, from our previous studies for having ideal solid fat contents whereas a random chemical esterification does not have (SFC) in the products, containing CLA. this specificity capability (5,6). In addition, enzyme- catalyzed reaction can occur in non-solvent system at a mild condition (7). Enzymatic esterification is used to Materials and Methods modify the physicochemical characteristics of oils and fats Materials and reagents Rice bran oil (RBO) was because the reaction can be used to produce modified fats obtained by Searim Company (Daejeon, Korea). Palm and oils by changing the fatty acids profile in the TAG stearin (PS) was generously supplied by CJ Co. (Seoul, molecules (8). For example, palm stearin (PS) was Korea). Conjugated linoleic acid (CLA) mixtures were esterified with vegetable oils to potentially promote the provided by Livemax Co. (Seongnam, Korea), which formation of desirable fats. Some researches have been contained 94.1% of CLA isomers. Lipozyme Thermomyces successfully attempted to prepare bakery fats with PS with lanuginosus (TL IM), which is a 1, 3-specific lipase other vegetable oils through lipase-catalyzed esterification granulated onto silica, was purchased from Novozymes A/ (9-11). S (Bagsvaerd, Denmark). According to the manufacturer Rice bran oil (RBO) has higher oxidative stability than specification, the specific activity of Lipozyme TL IM was soybean oil due to its lower level of linoleic and linolenic 175 IUN/g catalytic activity, having 0.54 g/mL bulk acid as well as the presence of relatively abundant amount density and 0.3-1.0 mm particle diameter. Acetone, hexane, and heptadecanoic acid were obtained from Sigma-Aldrich *Corresponding author: Tel: +82-42-821-6729; Fax: +82-42-822-6729 (St. Louis, MO, USA). E-mail: [email protected] Received October 16, 2008; Revised January 22, 2009; Fractionation of rice bran oil (RBO) The protocol of Accepted February 2, 2009 preparing fractionated S-RBO was similar to our previous

803 804 X. -M. Zhu et al. reports (17,18). RBO and acetone were mixed at volume 0.2% 2,7-dichlorofluorescein in methanol. Then, TAG ratio of 1:5 in a flask, maintaining at −16oC for 24 hr. The bands were scraped and methylated (18). A Hewlett- solid phase was collected, and removal of acetone was Packard 6890 Gas Chromatograph (GC, Avondale, PA, carried out by nitrogen blowing. USA) equipped with auto-injection and flame-ionization detection was used for fatty acid composition analysis. The Enzymatic esterification The substrates were prepared condition of GC was the same as previously described by mixing 0.6 g of S-RBO (Mw 861), 0.6 g of PS (Mw (17). The separation of fatty acid methyl esters was carried 850), and 0.4 g of CLA (Mw 280.4) with the mole ratio of out by a fused-silica capillary column (SP-WAX; 60 m 1:1:2 (S-RBO:PS:CLA) in a screw-capped test tube. ×0.25 mm; Supelco, Bellefonte, PA, USA). Besides, Another blend of 1:3:4 was prepared in the same way. heptadecanoic acid (C17:0; 50 µL of 1 mg/mL in hexane) After loading lipozyme TL IM (10% weight of total was added as an internal standard. substrate) into each blend, the esterification was conducted in a shaking water bath at 75ºC. The effect of time on Triacylglycerol (TAG) analysis The analysis of TAG incorporation of CLA and solid fat contents (SFC) was species of each reacted product was carried out by Yonglin ascertained by preparing a series of reactions in which 1, 3, SP930D HPLC (Yonglin, Anyang, Korea), which consisted 6, 12, 18, 24, 30, and 36 hr reaction. After each reaction by Sedex 75 evaporative light-scattering detector (Sedere, time passed, 1 g of reactant was dissolved in 5 mL of Alfortville, France), and Nova-Pak C18 column (150×3.9 hexane with 5 drops of phenolphthalein solution. Then, the mm, Waters, Milford, MA, USA). The separation was mixture was titrated with a 0.5 N KOH in 20% ethanol for obtained by gradient elution consisted of acetonitrile (A) deacidification. After titration, moderate hot water (30- and isopropanol/hexane (2:1, v/v) (B) at a flow rate of 1 40oC) was added and shaken vigorously. The hexane phase mL/min with the following profile: 0-44 min 20% B; 45- was then isolated. After passing through an anhydrous 50 min, 46% B; 51-58 min, 100% B. The TAG species sodium sulfate column the hexane phase was collected, and were identified with various TAG standards. hexane was removed under nitrogen gas with heating module (60oC) for obtaining each product. Solid fat contents (SFC) Melting thermograms of samples were obtained by differential scanning calorimeter (DSC) Fatty acid composition Thin layer chromatography (TLC) 2010 (TA Instruments Inc., New Castle, DE, USA). Purge was used for separation of the reactants on a silica gel 60 gas was nitrogen and the base line was obtained with an F254 plate (Merck KGaA, Darmstadt, Germany) developed empty aluminum pan. Each sample was weighed within with hexane/diethyl ether/acetic acid (50/50/1, v/v/v). The the range of 5.0 to 9.0 mg. For obtaining thermograms, triacylglycerol (TAG) bands were located by spraying with sample was heated at 80ºC and held for 10 min. Then,

Table 1. Fatty acid compositions (area%) of different time-reacted products1) of 1:1:2 and 1:3:4 Time (hr) 14:0 16:0 18:0 18:1 18:2 18:3 20:0 CLA ΣSFA ΣUFA RBO 0.3 19.4 1.7 45.9 32.1 0.6 ND ND 21.4 78.6 S-RBO 0.2 21.7 1.9 45.9 29.8 0.5 ND ND 23.8 76.2 PS 1.4 63.6 4.9 24.5 5.1 0.2 0.3 ND 70.2 29.8 CLA ND2) ND ND 3.9 2.0 ND ND 94.1 0 100 1 0.9 42.3 3.6 33.2 14.8 0.4 0.4 4.4 47.2 52.8 3 0.9 40.4 3.4 32.4 14.6 0.4 0.4 7.5 45.1 54.9 6 0.8 39.2 3.3 31.8 14.3 0.4 0.4 9.8 43.7 56.3 12 0.8 37.0 3.0 30.4 13.7 0.4 0.4 14.3 41.2 58.8 1:1:2 18 0.8 36.1 3.0 29.9 13.4 0.4 0.4 16.0 40.3 59.7 24 0.8 36.0 3.0 29.7 12.9 0.3 0.4 16.9 40.2 59.8 30 0.7 35.2 3.0 29.1 12.8 0.3 0.4 18.5 39.3 60.7 36 0.7 35.0 3.0 28.8 12.6 0.3 0.4 19.2 39.1 60.9 1 1.2 51.8 4.2 28.7 9.7 0.3 0.4 3.7 57.6 42.4 3 1.1 49.7 4.0 28.0 9.5 0.3 0.4 7.0 55.2 44.8 6 1.1 47.5 3.9 27.4 9.3 0.2 0.4 10.2 52.9 47.1 12 1.0 44.5 3.6 26.5 9.1 0.2 0.3 14.8 49.4 50.6 1:3:4 18 1.0 43.1 3.5 26.1 9.0 0.2 0.3 16.8 47.9 52.1 24 1.0 42.4 3.4 25.7 8.8 0.2 0.3 18.2 47.1 52.9 30 0.9 41.8 3.3 25.5 8.7 0.2 0.3 19.3 46.3 53.7 36 0.9 41.7 3.3 25.0 8.5 0.2 0.3 20.1 46.2 53.8 1)Enzymatically esterified solid fat with S-RBO:PS:CLA in a mole ratio of 1:1:2 and 1:3:4 catalyzed by lipozyme TL IM (10% weight) at 75ºC in a shaking water bath. RBO, rice bran oil; S-RBO, solid phase of fractionated rice bran oil; PS, palm stearin; CLA, conjugated linoleic acid; SFA, total saturated fatty acid; UFA, total unsaturated fatty acid. 2)Not detected. Production of Lipase-catalyzed Nutraceutical Fat 805

Table 2. Solid fat content (SFC, %) of different time-reacted products1) of 1:1:2 and 1:3:4 Time (hr) −10oC0oC5oC10oC15oC20oC25oC30oC35oC40oCSMP (oC) S-RBO 44.6 8.37 0.2 ND2) ND ND ND ND ND ND ND PS 99.6 92.7 86.3 78.4 74.0 73.6 71.7 63.0 53.2 43.3 52 1 75.9 60.3 51.0 47.0 46.2 39.1 30.8 23.7 16.9 9.5 44.5 3 80.6 62.3 52.5 46.1 45.2 37.4 27.7 19.5 11.7 3.6 38.0 6 77.0 58.7 49.7 42.0 41.1 32.9 23.3 15.33 7.6 1.1 34.0 12 75.6 58.7 48.8 38.0 36.5 27.7 19.1 11.6 4.26 0.8 33.5 1:1:2 18 73.6 57.5 46.7 35.7 33.6 24.6 16.3 9.2 2.1 0.1 29.0 24 72.0 47.0 30.1 21.2 17.4 13.7 6.4 1.0 0.3 0 27.5 30 73.3 45.5 29.1 20.5 16.8 13.4 6.3 0.9 0.2 0 26.0 36 74.7 45.0 27.2 18.8 15.4 12.8 6.2 1.4 0.1 0 26.0 1 89.9 75.9 68.6 62.1 61.6 59.1 49.5 40.5 31.5 22.3 47.0 3 88.0 72.6 65.3 57.8 56.8 54.5 44.4 35.1 26.0 16.5 42.5 6 90.6 74.7 67.2 58.6 56.6 53.4 41.3 31.0 21.4 11.4 41.5 12 88.3 71.9 65.1 55.78 52.9 48.4 36.3 26.1 16.5 6.76 40.0 1:3:4 18 87.9 71.4 65.4 55.0 52.1 46.3 33.7 23.2 13.3 3.6 37.5 24 89.9 73.4 67.5 56.2 52.7 46.9 33.9 22.9 12.5 2.5 36.9 30 87.8 71.7 66.5 54.2 50.7 46.1 33.6 22.8 12.2 2.0 36.0 36 86.4 70.7 65.7 54.1 50.52 45.1 32.66 22.0 11.8 1.9 36.0 1)Enzymatically esterified solid fat with S-RBO:PS:CLA (conjugated linoleic acid) in a mole ratio of 1:1:2 and 1:3:4 catalyzed by lipozyme TL IM (10% weight) at 75ºC in a shaking water bath. S-RBO, solid phase of fractionated rice bran oil; PS, palm stearin. SMP, slip melting point. 2)Not detected. sample was cooled down at a rate of 10ºC/min to −60ºC. 2. For S-RBO, the SFC at −10ºC was 44.6% while most After holding for 10 min, the melting curve was obtained phases were liquid at 5ºC (0.2% SFC). However, SFC (at by heating at a rate of 5ºC/min to 80ºC. −10ºC) of PS was 99.6%, indicating that most phases were solid. Even at 40ºC, SFC of PS was 43.3%. The reason Slip melting point (SMP) The determination of SMP could be contributed to high content of ΣSFA (70.2%) in was followed AOCS Official Method (19). All values were PS, while only 23.8% of ΣSFA was found in S-RBO obtained by average reading from 2 samples. (Table 1). After 36 hr reaction, the SFC (at 20ºC) of 1:1:2 product was 12.8% while the SFC of 1:3:4 product was Results and Discussion 45.1%. As predicated, high amount of PS increased SFC in the 1:3:4 product. Generally, the SFC at 20ºC determines Fatty acid composition The fatty acid compositions are the tendency of oil exudation, and a value of not less than listed in Table 1. Compared to original RBO, S-RBO 10% was recommended to prevent oiling off (22). contained higher amount of SFA because high melting TAG species were enriched after fractionation (16,20). Effect of esterification on SMP SMP is defined as the Similarly, 70.2% of ΣSFA presented in the PS. Thus the temperature at which the fat begins to rise in the capillary content of ΣSFA in blend of 1:3:4 (S-RBO:PS:CLA) tube. Table 2 shows the SMPs from the different time- were higher than that of 1:1:2 due to high amount of PS. reacted 1:1:2 and 1:3:4 products, showing 44.5-26.0ºC During the reaction of substrate blend with lipase, the and 47.0-36.0ºC, respectively. When the ratio of PS in the incorporation of CLA was gradually observed, resulting in substrate blend increased, SMP of the products generally decreasing amount of ΣSFA in the products. In addition, increased. Similar to the SFC results, the SMP also the effect of reaction time on the incorporation of CLA is decreased with prolonging the reaction time, resulting from shown in Table 1. The highest incorporation of CLA was the reduced ΣSFA mainly due to the incorporation of CLA observed after 36 hr reaction, showing that 19.2 and 20.1% in the products as shown in Table 1. of CLA in each 1:1:2 product and 1:3:4 product, respectively. On the contrary, only 4.4 and 3.7% of CLA Effect of esterification on TAG Figure 1 shows the were respectively found after 1 hr reaction. Therefore, it is newly produced TAG species and their composition after plausible that increasing reaction time allows the prolonged reaction. Generally, lipase TL IM-catalyzed esterification contact time between substrates and lipase, resulting in resulted in the rearrangement of fatty acids at the external increasing incorporation rate of CLA. (sn-1, 3 positions) glycerol backbone of TAG molecules while fatty acid at the internal position (sn-2) remained. In Effect of esterification on SFC The SFC was calculated Fig. 1, some peak areas of certain TAG species such as according to Lopez et al. (21) described. The SFC of S- PLO and PLP (P, palmitic acid; O, oleic acid; and L, RBO, PS, 1:1:2, and 1:3:4 product are presented in Table linoleic acid or CLA) increased in relative proportion while 806 X. -M. Zhu et al.

Fig. 1. HPLC chromatography of 1 hr 1:1:2 (S-RBO:PS:CLA; A) and 36 hr 1:1:2 (B) product. P, palmitic; O, oleic; L, linoleic and CLA.

POO, POP, and PPP decreased. In Table 2, changes of SFC With prolonging reaction time, the SFC was gradually were observed during 36 hr reaction. As a whole, the SFC reduced because of the increased incorporation of CLA in of 1:3:4 product at different reaction times was higher than each product. Even though similar CLA content was found those of 1:1:2 product due to the high amount of SFA from in each binary blends (1:1:2 and 1:3:4, S-RBO:PS:CLA) PS. When the reaction time increased, SFC of both 1:1:2 at different reaction times, more solid fat present in 1:3:4 and 1:3:4 product decreased at any temperature. For product due to higher amount of PS in the substrate blend. example, the SFC (at 20ºC) of 1:1:2 product was 39.1% after 1 hr reaction, which decreased to 12.8% after 36 hr reaction. One reason of decreased SFC by prolonging Acknowledgments reaction time is probably the increased incorporation of This research was supported by a grant from Korea Food CLA. As shown in Table 1, only 4.4% of CLA was found & Drug Administration in 2009. in 1:1:2 product after 1 hr reaction, and 19.2% of CLA was in the product after 36 hr reaction as well as high References melting (or hard) TAG species such as PPP and POP reduced. 1. Lee KN, Kritchevsky D, Pariza MW. Conjugated linoleic acid and In a conclusion, solid phase of solvent-fractionated RBO atherosclerosis in rabits. Atherosclerosis 108: 19-25 (1994) was used to prepare the solid fat in which CLA was 2. Chin SF, Storkson JM, Albright KJ, Cook ME, Pariza MW. Conjugated linoleic acid is a growth factor for rats as shown by intentionally incorporated. In this study, maximum content enhanced weight gain and improved feed efficiency. J. Nutr. 124: of CLA (19.2% in 1:1:2 product and 20.1% in 1:3:4 2344-2349 (1994) product) was obtained after 36 hr reaction, respectively. 3. O’Shea M, Bassaganya-Riera J, Mohede IC. Immunomodulatory Production of Lipase-catalyzed Nutraceutical Fat 807

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